Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

Abstract

Bovine liver cytochrome b (5) (cyt b (5)), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b (5) N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b (5) N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b (5) family, domestic silkworm cyt b (5) (DS cyt b (5)), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b (5) N57C, and Cys56, a naturally occurring cysteine in DS cyt b (5), have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b (5) maturation involves a b-type intermediate.

Peroxidase Activity of a c‐Type Cytochrome b5 in the Non‐Native State is Comparable to that of Native Peroxidases

“…Due to the spatial position, Cys57 forms a typical thioether linkage with a heme 4‐vinyl group, whereas Cys71 forms an unusual [heme‐CO‐CH 2 ‐S‐CH 2 ‐C α ] linkage with a heme 2‐vinyl group (Figure ). Molecular modeling indicated that covalent heme attachment to the polypeptide chain does not alter the overall structure of Cyt b 5 . The c ‐type Cyt b 5 , which differs from wild‐type (WT) Cyt b 5 by only two covalent linkages, provides an ideal protein model to investigate the structure and function of heme proteins in non‐native states and also the consequences of the heme‐to‐protein attachment.…”

“…Molecular modeling indicatedt hat covalent heme attachment to the polypeptide chain does not alter the overall structure of Cyt b 5 . [12] The c-type Cyt b 5 ,w hich differs from wild-type (WT) Cyt b 5 by only two covalent linkages, provides an ideal protein model to investigate the structure andf unction of heme proteins in non-native states and also the consequences of the heme-to-protein attachment. The double-mutant N57C/S71C Cyt b 5 was expressed and purified as previously described.…”

The broad diversity of heme-protein cross-links: An overview

“…Molecular modeling showed that a single thioether bond in N57C cyt b 5 does not alter the overall protein structure (Fig. 2D, [26]). The double thioether bonds in R98C/Y101C cyt b 562 , termed cyt cb 562 , were confirmed by an X-ray crystal structure (Fig.…”

“…With an emerging understanding, albeit not complete, of the diverse cross-links in natural heme proteins, most of these cross-links have been successfully recreated in natural or de novo proteins, such as Cys-heme [24,26,27,33,[41][42][43][44][45], Met-heme [53], His-heme [70][71][72], Trp/Tyr-heme [78] and Glu/Asp-heme [82,106,[108][109][110][111] cross-links. These achievements, in turn, have enhanced our knowledge of how heme-protein cross-link regulates the structure and function of heme proteins in nature.…”

“…Cyt b of cyt b 6 f; N57C cyt b 5 ; N57C/S71C cyt b 5 ; R98C/Y101C cyt b 562 ; S160C APX; H117C PCC 6803; Cyt P460; HAO Refs [21][22][23][24][25][26][27]33,44,68,129,130,133,134]. Vinyl group Cys C-S bond (heme-CO-CH 2 -S-CH 2 -C α ) Cyt rb 552 ; S71C cyt b 5 ; N57C/S71C cyt b 5 ;…”